9-cis retinoic acid inhibits inflammatory responses of adherent monocytes and increases their ability to induce classical monocyte migration.

Patients with vitamin A/retinol deficiency are shown to be prone to infections and to suffer from increased inflammation, effects which can be remedied by vitamin A supplements. We aimed to study how human monocytes from the peripheral venous blood of healthy donors acted within the initial hours after adherence and exposure to bacterial endotoxin in the presence or absence of the 9-cis-isomer of retinoic acid (9cisRA). We found that adherent human monocytes were dominated by the CD14dimCD16+ subtype. Pretreatment with 9cisRA for 1 h significantly decreased lipopolysaccharide (LPS)-induced mRNA expression and protein release of tumor necrosis factor (TNF)α, interleukin (IL)-6 and chemokine ligands (CCL)3 and CCL4. In contrast, treatment with 9cisRA rapidly enhanced the production of monocyte chemoattractive protein/CCL2. 9cisRA treatment also led to enhanced migration of classical CD14high monocytes in a transwell in vitro system. We conclude that 9cisRA treatment of human adherent monocytes attenuates the inflammatory responses to LPS and induces the attraction of classical monocytes, a feature which may help explain why supplements administered to vitamin A-deficient patients counteract inflammation and increases the ability to fight infections.

Liver X receptor protects against liver injury in sepsis caused by rodent cecal ligation and puncture.

BACKGROUND: Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). METHODS: Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. RESULTS: Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαß-deficient mice compared with wild-type and LXRß-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. CONCLUSIONS: Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.

Liver X receptor agonist GW3965 dose-dependently regulates lps-mediated liver injury and modulates posttranscriptional TNF-alpha production and p38 mitogen-activated protein kinase activation in liver macrophages.

Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells.

Immunmodulation of serum in orthopaedic trauma.

BACKGROUND: The aim of this study was to test the hypothesis that inhibitory substances circulating in the patient's serum after trauma might impair leukocyte function by evaluating the effect of such serum on cytokine release in a whole blood model. METHODS: Hip replacement surgery was considered a standardized musculoskeletal trauma, and seven women and three men undergoing elective total hip replacement were included in the study. Ex vivo lipopolysaccharide (LPS) and peptidoglycan (PepG) induced tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL-10) releases were measured in whole blood sampled preoperatively and added serum taken before, at the end of operation and at postoperative day 1 and 6. Saline was used as negative control to serum. RESULTS: LPS and PepG induced a significant release of TNF-alpha and IL-10 in whole blood. Addition of preoperative serum, postoperative serum or day 1 postoperative serum did not alter the LPS-induced release of TNF-alpha as compared with saline control. Addition of preoperative serum significantly increased the PepG-induced release of TNF-alpha as compared with saline control (p = 0.011). This increase was not significantly changed with addition of postoperative serum or day 1 postoperative serum. When serum from postoperative day 6 was added, both LPS and PepG induced expression of TNF-alpha was significantly reduced as compared with preoperative serum (p = 0.018 and 0.008, respectively). Preoperative serum also increased the PepG induced expression of IL-10 (p = 0.007) in relation to saline control, and this increase was not significantly changed by addition of postoperative serum or day 1 and day 6 postoperative serum. Neither of the serum samples altered the LPS induced expression of IL-10 as compared with saline control (p = 0.212). CONCLUSION: Our data show that in trauma patients, serum expresses activity that inhibits LPS and PepG induced release of TNF-alpha in a whole blood model, and our study, then, corroborates the hypothesis that inhibitory substances circulating in the patients' serum after trauma impair leukocyte function.

Systemic administration of enamel matrix derivative to lipopolysaccharide-challenged pigs: effects on the inflammatory response.

BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.

Differences in LPS and PepG induced release of inflammatory cytokines in orthopedic trauma.

Trauma is associated with immune paresis which may predispose to postoperative sepsis. We characterized the ex vivo cytokine responses to bacterial cell wall components in whole blood from 8 patients undergoing a major musculoskeletal trauma in the form of total hip replacement. Preoperatively, at the end of operation, and at days 1 and 6 postoperatively, patient blood was obtained, anticoagulated, and incubated at 37 degrees C in the presence of peptidoglycan (PepG) or lipopolysaccharide (LPS). Plasma cytokines were measured by enzyme immunoassay. The numbers of leucocytes, monocytes, and neutrophils were unchanged at the end of surgery, while there were significant increases at postoperative days 1 and 6. We observed significant reductions in tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10) responses to PepG at the end of surgery, which was disproportional to the nonsignificant reductions in circulating monocytes, suggesting a functional suppression. However, at postoperative day 1 the responses were recovered. There were no significant changes in responses of TNF-alpha to LPS stimulation at the end of surgery, while there were significant depressions at postoperative days 1 and 6. The expression of IL-10 was significantly depressed at the end of surgery and at day 6. There were modest changes in PepG- and LPS-induced expression of interleukin 8 (IL-8) during the experiments. On the basis of these observations, we conclude that a musculoskeletal trauma is associated with reduced expression of TNF-alpha and IL-10 by whole blood leucocytes when exposed to endotoxin, but there is a difference between gram-positive endotoxin (PepG) and gram-negative endotoxin (LPS).

Liver X receptor is a key regulator of cytokine release in human monocytes.

Aberrant regulation of innate immune responses and uncontrolled cytokine bursts are hallmarks of sepsis and endotoxemia. Activation of the nuclear liver X receptor (LXR) was recently demonstrated to suppress inflammatory genes. Our aim was to investigate the expression of LXR in human monocytes under normal and endotoxemic conditions and to study the influence of LXR activation on endotoxin-induced cytokine synthesis and release. Adherent human monocytes or whole blood were pretreated with a synthetic LXR agonist (3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid) and subsequently challenged with LPS (from Escherichia coli) or peptidoglycan (from Staphylococcus aureus). Cytokine release was assessed by a Multiplex antibody bead kit, and cytokine mRNA levels were measured by real-time reverse-transcriptase-polymerase chain reaction. We found that LXRalpha mRNA was up-regulated in CD14+ monocytes in LPS-challenged blood, whereas LXRbeta mRNA was not altered. Addition of 3-{3-[(2-chloro-3-trifluoromethyl-benzyl)-(2,2-diphenyl-ethyl)-amino]-propoxy}-phenyl)-acetic acid to monocytes suppressed the LPS-induced release of IL-1beta, IL-6, IL-8, IL-10, IL-12p40, TMF-alpha, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, and monocyte chemoattractant protein 1 in a concentration-dependent manner. Surprisingly, an accompanying decrease in cytokine mRNA accumulation was not observed. The suppressed cytokine release could not be explained by a diminished transport of mRNA out of the nucleus or a decreased secretion of cytokines. We propose that LXR is a key regulator of cytokine release in LPS-challenged human monocytes, possibly by interfering with translational events.

Activation of cytokine synthesis by systemic infusions of lipopolysaccharide and peptidoglycan in a porcine model in vivo and in vitro.

BACKGROUND: The incidence of gram-positive and mixed bacterial infections in surgical patients has increased, and there has been an alarming rise in the number of drug-resistant bacteria. Peptidoglycan (PepG) is a cell wall component of gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. The systemic effects of PepG on inflammation have not been studied in a large animal model. METHODS: Anesthetized pigs were subjected to 8-h continuous intravenous infusions of lipopolysaccharide (LPS) (4 mcg/kg/h), PepG (40 mcg/kg/h), LPS plus PepG, or saline. The concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and IL-10 were measured in the plasma prior to infusion (time 0) and thereafter every second hour until the end of the experiments. Heparinized whole blood samples drawn at time 0 and after a 6-h infusion of LPS or PepG were incubated ex vivo with PepG (10 mcg/mL), LPS (10 ng/mL), or a combination of PepG and LPS to study the immunologic consequences of systemic inflammation. Concentrations of TNF-alpha, IL-8, and IL-1beta were measured in the supernatant liquids. RESULTS: In vivo, there was transient upregulation of TNF-alpha after infusion of LPS, PepG, or the combination. Interleukin-6 and IL-8 were upregulated by LPS but not by PepG. In vitro studies of whole blood obtained at time 0 revealed a synergistic effect of LPS and PepG on the release of TNF-alpha. Incubation of whole blood obtained after 6 h of infusion of LPS or PepG revealed tolerance and cross-tolerance between the two bacterial components in the induction of TNF-alpha, IL-8, and IL-1beta. CONCLUSIONS: Peptidoglycan is a potent inducer of TNF-alpha in this large animal model. Peptidoglycan and LPS synergized to increase the formation of the proinflammatory cytokine TNF-alpha. The study demonstrates for the first time the development of tolerance and cross-tolerance between LPS and PepG in a large animal model. These phenomena could be of importance for the signs and symptoms of sepsis.

Postoperative serum attenuates LPS-induced release of TNF-alpha in orthopaedic surgery.

Studies with ex vivo stimulation of whole blood samples from injured patients have revealed a diminished production capacity for a broad range of secretory products, including inflammatory cytokines. Recent interest has focused on the release of mediators in serum that depress the cell-mediated immune response following trauma. The involvement of the lipid mediator prostaglandin E2 (PGE2) has been assumed because it is a potent endogenous immunosuppressor. In the present study, we tested the hypothesis that inhibitory substances circulating in the patient's serum after a major musculoskeletal trauma might impair leukocyte function by evaluating the effect of such serum on cytokine release in a whole blood model. Six females and three males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-alpha and IL-10 were measured in whole blood sampled preoperatively and added serum taken before, at the end of operation, and at postoperative days 1 and 6 with saline as negative control. LPS induced significant releases of TNF-alpha and IL-10 in whole blood. Addition of preoperative, postoperative, and day-1 postoperative serum did not alter the LPS-induced release of TNF-alpha as compared to saline. In the presence of serum from postoperative day 6, however, the expression of TNF-alpha was significantly reduced as compared to saline and preoperative serum (p = 0.021 and 0.008, respectively). Neither of the serum samples altered the release of IL-10. PGE2 was significantly (p = 0.008) increased in serum at postoperative day 6 as compared to preoperative levels. In conclusion, these data show that at day 6 after major orthopaedic surgery, the patient serum contained activity that inhibited ex vivo LPS-induced TNF-alpha release. The potent TNF-alpha inhibitory activity found at day 6 after injury correlated with increased levels of PGE2 and indicates cell-mediated hyporesponsiveness to a second stimulus.

Alterations in inflammatory capacity and TLR expression on monocytes and neutrophils after cardiopulmonary bypass.

Cardiopulmonary bypass (CPB) is associated with immune paresis, which predisposes to the development of postoperative sepsis. The aims of this study were to characterize the ex vivo cytokine responses to bacterial cell wall components in whole blood from patients undergoing CPB and to determine whether altered leukocyte expression of Toll-like receptors (TLRs) is involved in immune paresis after CPB. We recruited 6 patients undergoing routine cardiac surgery with CPB. Preoperatively, at the end of CPB and 20 h later, blood was obtained, anticoagulated, and leukocyte surface expression of CD14, TLR2, and TLR4 was quantified by flow cytometry. In addition, blood was incubated at 37 degrees C in the presence of peptidoglycan (PepG) and/or lipopolysaccharide (LPS), and plasma cytokines were measured by enzyme immunoassay. At the end of CPB, ex vivo production of tumor necrosis factor alpha, interleukin (IL) 1beta, IL-8, and IL-10 in response to PepG or LPS was virtually abolished (P < 0.05). The following day, there was recovery of all cytokine responses to PepG. Tumor necrosis factor alpha and IL-1beta responses to LPS partially recovered, whereas IL-8 and IL-10 responses recovered. At the end of CPB, there was more than 50% reduction in neutrophil TLR2 and TLR4 expression (P < 0.05), with recovery to baseline the following day. There was a 29% reduction in monocyte TLR4 expression at the end of CPB (P < 0.05) and more than 120% increase in monocyte TLR2 and 4 expression the following day (P < 0.05). In conclusion, reduced ex vivo production of cytokines cannot be fully accounted for by downregulation of TLR expression, although receptor upregulation may contribute to the later recovery of responsiveness.

Lipopolysaccharide attenuates mRNA levels of several adenylyl cyclase isoforms in vivo.

Signals that elevate intracellular levels of cyclic adenosine monophosphate (cAMP) are among the factors that control lipopolysaccharide (LPS)-mediated inflammatory mediator production by macrophages. cAMP signaling is also involved in maintaining body functions that are commonly impaired in sepsis, including the endothelial cell barrier function and heart function. Several agents successfully used for sepsis intervention target cAMP signaling, and it was recently shown that liver and lung may be protected from inflammation injury by cAMP-elevating phosphodiesterase inhibitors. Here, we show that LPS attenuates adenylyl cyclase (AC) mRNA levels in liver, lung, heart, spleen and kidney in an animal model of endotoxemia, and in macrophages from liver and lung. In particular, AC5, AC6, AC7 and AC9 mRNA were reduced in most tissues examined and in tissue macrophages. In Kupffer cells, prostaglandin E2-mediated cAMP production was inhibited by LPS treatment. The reduction in AC mRNA by LPS would be expected to lead to a lowered potential for cAMP production in most organs, and in particular, changes in AC6 mRNA may affect endothelial cell barrier function and heart function. In contrast, AC4 mRNA was elevated in heart and lung. The present work indicates a possible mechanism for LPS-mediated alteration of cAMP signaling in vivo.

Staphylococcus aureus peptidoglycan impairs fracture healing: an experimental study in rats.

Staphylococcus aureus is the common organism causing musculoskeletal infections. Staphylococcus aureus peptidoglycan (SaPG) has been identified to increase the acute inflammatory response to wounding, increase reparative granulation tissue, and improve healing. The healing of bone fractures is a balanced process of granulation tissue that is calcified to obtain increasing stability. By increasing reparative collagen accumulation, however, SaPG may induce a shift towards immature fibrous callus production. Therefore, it was our hypothesis that SaPG would impair bone healing after fracture. In three groups, each of nine rats, a mid-diaphyseal osteotomy/fracture of the femoral bone was performed and then nailed. In one group of animals, SaPG was applied locally at the fracture site, and in another group SaPG was applied intraperitoneally (systemically). Control littermate received saline. The animals were sacrificed after 6 weeks, and the mechanical characteristics of the healing osteotomies were evaluated. We found that application of SaPG locally induced a hypertrophic and immature callus as evaluated by callus production, by bone mineral content and density, and by bending moment and rigidity. In the rats given SaPG intraperitoneally, bone healing went uneventful compared to the control rats. Collectively, these data show that SaPG induces an alteration in the normal bone healing response towards a less calcified callus production.

Mediator responses in surgical infections.

BACKGROUND: Surgical infections, and sepsis in particular, are characterized by extensive release of mediators. Our laboratories have been interested in understanding how these substances contribute to morbidity and mortality during various stages of surgical infections in order to develop new and more effective therapeutics and treatment strategies. METHODS: In a series of in vitro studies, human plasma was exposed to lipopolysaccharide (LPS), and whole blood was treated with peptidoglycan from Staphylococcus aureus. The activity of peptidoglycan also was studied in the rat, and LPS infusion was tested in dog and pig models. In a clinical study, the relation of serum LPS to multiple organ dysfunction and failure was studied in patients in the surgical intensive care unit. RESULTS: Exposure of plasma to LPS led to formation of bradykinin, activation of the plasma kallikrein-kinin system, and reduction of kallikrein inhibitor capacity. The coagulation, fibrinolysis, and complement cascades were activated. Peptidoglycan caused rapid release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 from macrophages and activation of the genes encoding pro-inflammatory and anti-inflammatory cytokines. In the rat, peptidoglycan induced cytokine release, caused liver and kidney dysfunction, and induced matrix metalloproteinase-9 (MMP-9) activity in the liver and lung. In the dog and pig, LPS caused substantial activation of plasma proteases. Clinically, a finding of LPS in the plasma was associated with multiple organ dysfunction and failure. These patients also revealed substantial activation of the plasma cascade systems, as well as systemic cytokine release. CONCLUSION: On the basis of these observations, we developed a monitoring system to recognize early signs of infection.

The role of the surgeon as intensivist: an international perspective.

PURPOSE OF REVIEW: Critical care is a young specialty. It emerged less than 50 years ago in response to new technologies that could prolong the survival of patients who previously would have died. The diseases that posed this threat to life were varied and the original practitioners of critical care came from a variety of medical backgrounds, and created a multidisciplinary specialty. As it continues to evolve, however, the participation and influence of surgeons is waning. CONTENT: We have sought the perspectives of an international group of intensivists on the role of surgeons in the future of critical care. These perspectives are varied. Surgeons play the largest role in North American critical care, although even here that role is diminishing, and new models of practice are needed. Surgical involvement is even less in Japan, Europe, and South America. The important role of the surgical perspective in differentiating heroic from futile intervention is underlined by Alsanea from Saudi Arabia. CONCLUSIONS: Surgical involvement in the practice of critical care is declining around the world. The reasons are complex, and include competing clinical imperatives, economics, and the demands of training programs. New models are needed to revitalize critical care as a multidisciplinary specialty.

Thalidomide attenuates the development of fibrosis during post-infarction myocardial remodelling in rats.

BACKGROUND: Inflammation plays a pathogenic role in the development of heart failure (HF). The aim of this study was to examine the effect of treatment with the immunomodulating drug thalidomide in a rat model for post-myocardial infarction (MI) HF. METHODS: Rats were subjected to MI by left coronary artery ligation or sham-operated. Seven days after surgical intervention rats were randomised to treatment with thalidomide or vehicle for 8 weeks. RESULTS: Our main findings were: (i) thalidomide treatment did not affect cardiac function or the hypertrophic response, as determined by haemodynamic measurements and heart chamber weights, respectively. (ii) HF rats treated with thalidomide had a minor reduction in septum and relative wall thickness (p<0.05), indicating an anti-remodelling effect. (iii) Thalidomide appeared to have immunostimulatory effects on the myocardium as evident by increased MIP-1alpha gene expression (p<0.05). (iv) Treating HF rats with thalidomide reduced myocardial collagen content, as assessed by markedly decreased levels of hydroxyproline ( approximately 40% reduction; p<0.05), accompanied by lower TGF-beta(1) gene expression (p<0.05). CONCLUSION: Although thalidomide had no effect on cardiac function, our results suggest that intervention with thalidomide may have beneficial effects in post-MI HF by attenuating collagen accumulation and development of myocardial fibrosis.

Peptidoglycan--an endotoxin in its own right?

Studies aimed at dissecting the complex pathophysiology of sepsis with multiple organ failure have traditionally focused on lipopolysaccharide of gram-negative bacteria, which is widely regarded as the classical endotoxin. However, gram-positive sepsis now accounts for up to 50% of all cases, calling for a shift of focus. Peptidoglycan (PepG) is the major cell wall component of gram-positive bacteria and has been increasingly recognized as an important proinflammatory molecule. During gram-positive infections, PepG reaches the circulation by bacterial breakdown or translocation from the intestine. Administration of PepG induces all the classical features of infectious illness and endotoxemia and may cause systemic inflammation with organ failure in animal models. Its potency, however, is crucially dependent on various features of its complex structure. PepG interacts with the innate immune system through receptors mainly expressed on monocytes/macrophages but may induce inflammatory changes in other cell types as well. Among the most extensively studied receptor systems are the nucleotide-binding oligomerization domains, the toll-like receptors, and the PepG recognition proteins. Based on the current available literature, we would like to propose that PepG must be regarded as an endotoxin in its own right and to encourage further work in the field of PepG signaling.

Activation of the liver X receptor protects against hepatic injury in endotoxemia by suppressing Kupffer cell activation.

Recent reports have demonstrated that liver X receptors (LXRs) of the nuclear receptor family have anti-inflammatory effects on macrophages. Here we examine whether activation of LXR by the synthetic agonist GW3965 can ameliorate the liver injury/dysfunction caused by endotoxins in the rat. Male Wistar rats received GW3965 (0.3 mg/kg) or vehicle (50% dimethyl sulfoxide) 30 min before coadministration of lipopolysaccharide (LPS, 5 mg/kg i.v.) and peptidoglycan (1 mg/kg i.v.). Treatment with GW3965 attenuated the increase in the plasma levels of alanine aminotransferase and bilirubin (markers of liver injury/dysfunction) as well as the focal hepatocyte necrosis (histology) caused by coadministration of LPS and peptidoglycan. This protective effect of GW3965 treatment was associated with reduced infiltration of mast cells in the liver (histopathology) and reduced gene expression of the chemokines eotaxins 1 and 2, whereas MIP-2 mRNA levels were not affected. Plasma levels of tumor necrosis factor alpha and prostaglandin E2 were significantly attenuated by GW3965, whereas plasma interleukins 6 and 10 were not altered. High expression of LXRalpha mRNA was observed in Kupffer cell cultures, suggesting that Kupffer cells are targets of GW3965. Subsequent in vitro studies in Kupffer cells demonstrated that exposure to GW3965 attenuated the LPS-induced release of tumor necrosis factor alpha and prostaglandin E2 in a dose-dependent manner. In conclusion, this study demonstrates that activation of LXR by GW3965 protects against liver injury and dysfunction in a rat model of endotoxemia, in part by exerting an anti-inflammatory effect on Kupffer cells.

Effects of forskolin on Kupffer cell production of interleukin-10 and tumor necrosis factor alpha differ from those of endogenous adenylyl cyclase activators: possible role for adenylyl cyclase 9.

Proinflammatory cytokines like tumor necrosis factor alpha (TNF-alpha) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-alpha expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and beta-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 microg/ml) caused attenuated TNF-alpha levels in culture medium (forskolin/isoproterenol, P < or = 0.05; prostaglandin E2, P < or = 0.01). Forskolin also reduced IL-10 mRNA and protein (P < or = 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P < or = 0.05). We conclude that regulation of TNF-alpha and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2 differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9.